Generally this invention relates to the field of detecting the use of controlled substances such as illicit drugs and the like. More specifically, the invention involves the field of Raman spectroscopy to accomplishing detection and the sub-field known as Surface Enhanced Raman Scattering. In a less focused sense, this invention relates to a method of detecting substances through the use of a coated surface and spectroscopic techniques. The invention also covers the use of a new coating with a tethered reactive species that chemically binds to an analyte. Through this new technique, the analyte is then spectroscopically analyzed as part of a new chemical species which is the reaction product of the analyte and the reactive species.
The field of sensing controlled substances is an area which has evolved primarily for the public good. As practically everyone knows, drug and alcohol abuse are significant problems for society. In fact, in 1998 the United Nations conducted an international anti-drug conference involving over one hundred nations which are facing this societal challenge. As society attempts to address this problem it has turned to increasingly sophisticated technical analysis to counter the abuser""s attempt to hide either the controlled substance or its use. Naturally, the principles of analytical chemistry have been relied upon for their ability to not only detect but to discriminate the presence of a controlled substance in minute amounts. Unfortunately at its present state, the field of detecting controlled substances still has difficulty in both sensing and discriminating the existence of some substances as well as in avoiding false positive indications. This invention provides a solution that greatly expands the techniques and accuracy available for a variety of substances. It also provides a framework under which practical advantages can now be achieved. These advantages range from the seemingly simple ability to provide a single sensor for a variety of drugs as well as the ability to now be able to discriminate between controlled substances and some chemically similar uncontrolled substances. This latter aspect can be significant because in a variety of applications such as Olympic drug testing and the like, it has become difficult to accurately sense and distinguish the difference between certain substances which are legally available for use and those which are truly illegal substances. In a broader sense, the invention also provides an expansion to the principles of analytical chemistry since it may be applied in other areas as well.
The field of analytical chemistry dates back at least to Pliny the Elder (AD 23-79) who first described the use of an extract from gallnuts that turns black in the presence of iron sulfate. This allowed him to determine if copper sulfate was contaminated with iron sulfate. This simple concept of chemical analysis has grown into analytical chemistry which is one of the four disciplines of modern chemistry. Analytical chemistry encompasses a variety of fields such as clinical chemistry, environmental chemistry, geochemistry, and forensic chemistry. The techniques of analytical chemistry have grown from the simple wet chemical analysis discovered by Pliny the Elder to very sophisticated instrumental methods. Early analytical chemistry relied on visual observation of color changes or the precipitation of a compound to quantitate materials. This meant that the sensitivity was often limited to the visual acuity of the chemist. Instruments have largely replaced these visual techniques, since it is possible to electronically detect changes in light intensity and wavelength with vastly superior sensitivity.
The electronic detection of changes in light intensity and its separation into different wavelengths is the basis of the field of analytical spectroscopy. This is an area in which some type of analyte, namely, some substance or chemical which is desired to be studied, is exposed to some wavelength of energy. This wavelength may be a singular wavelength such as a laser often provides, or it may be many different wavelengths. To provide the information desired, the analyte then causes some type of change in that incident energy and thus results in some type of change in intensity of at least one wavelength of energy which is characteristic of the analyte. Thus the wavelength of energy to which the analyte is exposed and the changed signal resulting usually vary. Naturally, the incident energy may be present in a variety of forms as those aware of the wave-particle duality may easily understand. Essentially, however, all that spectroscopy involves is an incident wavelength which is somehow affected by an analyte to result in a changed signal. This signal may be a singular wavelength or may be a broad spectrum of wavelengths of emission or adsorption. Thus, xe2x80x9cspectroscopyxe2x80x9d as intended here is not intended to be limited to only some type of slit-based instrument, but rather is intended to fully encompass the areas of analytical chemistry in which changes in wavelengths of energy are studied to gain information with regards to an analyte. Conversely, it should be understood that other fields or areas of study which do not involve changed wavelengths have not been viewed as particularly relevant to this field. For example, the areas of chromatography and the like which act to separate substances, immunoassays which transiently bind substances for nonspectroscopic purposes, and the like, have not been viewed as particularly relevant to the fields in which this invention relates.
As mentioned earlier, the field of sensing controlled substances faces a variety of limitations. These range from imperfect discrimination (such as in the Olympic drug testing scenarios) to practical challenges such as the need to have different tests for different substances. When considering spectroscopic techniques, great improvement has occurred through the introduction of a technique known as Raman spectroscopy. Raman spectroscopy was discovered by Sir Chandrasekhara Venkata Raman in the early 1900s who found that different chemicals sometimes caused unique scattering of an incident wavelength of energy. Since the scattering was largely unique to each chemical, the analysis of the specific scattering thus provided information from which specific chemical detection and identification could be achieved. Unfortunately, limits remained even with the introduction of Raman spectroscopy.
In 1976, a new spectroscopic technique was discovered that is sensitive to interfaces. This technique has been coined Surface Enhanced Raman Scattering (SERS). SERS tends to give large enhancements of Raman scattering in the presence of certain prepared metallic surfaces. The SERS technique has been applied to a variety of problemsxe2x80x94not only those associated with analytical chemistryxe2x80x94and more recently has been the subject of several publications and patents for analytical chemistry. This technique generally involved some type of attachment of an analyte to a metal surface or to a coating on a metal surface such as gold or silver.
Even in the broader area of general analytical chemistry, initially, the coatings were not tethered to the metal surface. In U.S. Pat. No. 5,326,211 relating to analytical chemistry in general, Carron and Mullen showed that it was possible to coat a surface with a dye that had complexed with a metal ion to serve as a metal ion detector. Again in the broader field of analytical chemistry, Angel was awarded an early patent, U.S. Pat. No. 4,781,458 for the determination of analytes adsorbed directly to metals surface or partitioned onto a coating. Even more recently some publications by Carron and U.S. Pat. No. 5,327,211 disclosed the use of SERS with coatings that contain thiols to tether the coatings to a silver surface. That disclosure specifically addresses the use of SERS coatings on a fiber optic to allow for remote sensing. Carron, et. al, has demonstrated that the coatings mimic separation science coatings and also serve to stabilize the SERS substrate to give it longevity. The coatings can also provide an internal standard that allows one to use relative intensities to determine a calibration that can be used to find the concentration of the analyte.
The general techniques of SERS has been well established and is discussed to some degree not only in the above general analytical chemistry references, but also in a variety of references ranging from text books to additional patents such as U.S. Pat. No. 5,693,152 to Carron, U.S. Pat. No. 5,255,067 to Carrabba, and U.S. Pat. Nos. 5,266,498, 5,376,556, and 5,567,628 to Tarcha in the immunoassay field. To the extent necessary, each of these references is hereby incorporated by reference to provide additional understandings as they relate to the Raman and SERS techniques generally.
One of the perspectives that has evolved to those focusing on these techniques has been a perspective that suggests that it may be more appropriate to avoid changing any structures or spectral characteristics of an analyte during its spectroscopic analysis. This perspective seems rooted in the understanding that, naturally, if one wants to detect the analyte itself, it would be better not to alter the analyte or controlled substance. Thus, the coatings developed to date for analytical applications of SERS have been coatings that weakly interact with the analyte to produce a reversible measurement of the analyte concentration. These include alkylthiols that mimic the nonpolar coatings used in reverse phase HPLC, pH and metal sensitive coatings that mimic ion chromatography, and oxide coatings that mimic normal phase chromatography.
By weakly interacting with the analyte to produce a measurable spectrum, the goal of seeing only the analyte spectrum had been satisfied. As a result, the techniques of Raman spectro-scopy and specifically that of Surface Enhanced Raman Scattering, had to some degree been viewed as limited since the surfaces typically used in SERS have been the relatively unreactive substances of gold and silver. Since these substances can cause the desired weak interactions in only a few situations, these techniques, while powerful for certain analytes, had not been as greatly exploited as possible in the field of detection of controlled substances. In essence, since a goal was that the surface itself created some sort of weak link with the controlled substance, and since not many controlled substances would establish an appropriate link with the typically required gold or silver surfaces, these techniques were often not viewed as particularly appropriate to the field of detection and analysis of controlled substances or specific substances and the like. The techniques were also viewed as somewhat limited themselves because it seemed that they could really only be used for those specific types of analytes that happened to bond appropriately to the required surface. Although eventually different sample surfaces did exist for certain different analytes, generally the selections were so limited that the techniques were perhaps underutilized.
One example of efforts to alter the surface involved in the SERS technique is disclosed in U.S. Pat. No. 5,693,152 to Carron, one of the present inventors. In that patent, Carron disclosed a technique to modify the surface enhanced Raman scattering (SERS) detector by applying a stabilizing coating on the SERS surface. The coating applied would reproduce or mimic the specific separation procedure being utilized thus this method could be used universally for all types of analytes and separation methods. Similar to the invention disclosed in a patent by Carrabba et al (U.S. Pat. No. 5,255,067), a roughened surface substrate was used to improve SERS detection efficiency in gas chromatography. However, although the coating disclosed in ""152 patent can locally increase the analyte concentration and improve linking affinity between the coating and the analyte, the attachment of these analytes to the coatings are still through weaker linking mechanisms, primarily by means of adsorption or other weak forces.
Similarly, other, perhaps unrelated fields have seen efforts to alter binding sites in the immunoassay area. As mentioned earlier, the use of SERS technology for immunoassays has been disclosed by Tarcha et al. In U.S. Pat. Nos. 5,266,498, 5,376,556, and 5,567,628. In their disclosures the authors designed a Raman active reporter which is bound to a specific binding member. However, this is not only an immunoassay method, it involves attachment of an analyte to a binding member through weaker techniques and thus even though it is in an unrelated area, seems to show the pervasiveness of the perspectives and attitudes of those involved in Raman spectroscopy.
In the focused field of detecting controlled substances, however, the problems seem even more acute. Those involved in sensing controlled substances (and other, even uncontrolled substances) have faced problems with the discrimination abilities between substances. The problems for professional athletes who may have taken some type cold medicine prior to their participation in the Olympic games has been highly popularized. From one perspective this problem may be viewed as a simple byproduct of the inability of the analytical techniques to adequately discriminate between a legal cold medicine and the remnants as a result of the use of an illegal drug.
Another problem which is the fact that it is desirable to sense controlled substances in extremely low concentrations. Again, the seemingly irresolute requirement that the analyte or controlled substance only weakly binds with the spectroscopic surface has made it challenging to sense such low concentrations because the equilibrium values are often such that the analyte would desorb from the surface and not permit a sufficient build up to facilitate high quality detection. Further, a problem has existed in instances in which it was desired to not only detect but to quantify the presence of either some controlled substance or more generally an analyte, over a broad ranges of concentrations. In those instances in which such spectroscopic techniques were found to apply, it was often the case that the ranges over which a specific sensor could be used made it practically difficult to achieve the technique.
Furthermore, because there are a great variety of controlled substances, one of the problems has been in the ability to use the specific spectroscopic technique to sense not only one specific type of controlled substance but to sense a great variety of substances. Not only have there existed limitations on which specific substances interact appropriately with which specific sensors, but there have existed seemingly fundamental limitations such that certain substances could not be analyzed through Raman techniques. Naturally, while many of these problems are particularly acute in the focused field of sensing controlled substances, it can be easily understood that these problems also applied to more general applications of spectroscopic techniques as well.
One way in which the apparent requirement of a weak interaction has resulted in a difficulty is the practical fact that interfering substances are often present. While it would be desirable to remove these interfering substances, the weak interaction has made it more difficult to remove the interfering substances without also removing some analyte as well. Even the improved SERS coating disclosed in U.S. Pat. No. 5,693,152 highlights that this problem continued unsolved. Essentially, the problem was that while such a SERS coating could specifically adsorb material from the matrix, there were no ways of washing the surface to remove the interferences either unavoidably or practically present in the matrix. Furthermore, since the interaction of the analyte with the surface was often viewed as necessarily reversible, it was implied that the interaction needed to be weak. Physically, this often meant that only a small amount of the analyte tended to interact with the surface or could be detected in a given instance.
Thus, in the field of sensing controlled substances, there has been a long felt but unsatisfied need for a detection technique which could be applied to a great number of controlled substances and which could also provide a higher degree of discrimination between such substances especially those which were not considered illegal. Even though those skilled in the field of sensing controlled substances appreciated this desire, they seemed not to have fully appreciated the nature of the problem in that their perspective was driven by certain preconceptions which actually limited their ability to solve the problems with which they were faced. To some degree their substantial attempts failed to fill the need either because their field did not require the basic physical understanding of the phenomenon or they simply assumed that existing techniques could not be adapted to their unique needs. For this reason, it appears that those skilled in the art to some degree actually taught away from the direction in which the present inventors went.
As related to the broader field of analytical chemistry in general, it appears that similar perspectives also apply. For instance, while those in the field of analytical chemistry in general well appreciated that it was desirable to apply sensitive techniques such as Raman spectroscopy and the SERS techniques to a greater variety of substances, their apparent tendency was to approach the problems from the perspective of seeing primarily the analyte as opposed to some altered by-product. Again, while they had expended substantial efforts to expand the applicability of the techniques, their focus toward physisorption perhaps showed that they did not fully appreciate the nature of the problem. They seemed thus to teach away from the direction in which the present inventors went pursuing avenues that in hindsight might be viewed as based on misperceptions to some degree. Not only were these misperceptions fostered by the initial desire to sense an unaltered analyte, but they also were fostered by attitudes which seemed to suggest that reversible and remote sensing arrangements were required for some practical reason. Thus, to some degree the present invention might even be characterized as unexpected in the sense that it proposes techniques and substances which, prior to this invention, were not just deemed suboptimal but rather were viewed as contrary to the goals typically considered and the results typically desired.
In addition to the aspect of applying analytical techniques to a greater variety of substances, those generally applying the techniques had long felt a need for an ability to apply those techniques to greater ranges of concentrations and to greater varieties of chemicals during one analysis event. Even though a desire existed, they may not have fully appreciated that the problem and solution lay not in sample preparation or non-spectroscopic techniques, but in adjustments to the analytical technique itself or to the specific sensors involved in the analytical technique. Efforts focussed in a direction different from those of the present inventors may have been due to the fact that those involved did not to some degree fully appreciate that sensor chemistry could offer the needed advances and simplifications.
To address these and other problems, the present invention involves techniques and devices which offer improved spectroscopic analysis capabilities. As applied to the field of sensing controlled substances, the invention involves the creation of a unique surface coating such as a diazonium or other type of coating which interacts with the substance in a wholly different manner. Rather than forming the weak interactions that were previously viewed as perhaps required, the invention creates a whole different type of interaction, namely a full reaction such as in the formation of a covalent bond to produce an entirely different species, termed here in adduct. Since in most instances the formation of this covalent bond is irreversible, the rate of loss of the controlled substance or other analyte from the surface will be approximately zero. As a result, washing or other steps to remove interference substances can now be accomplished. Thus, as a result, the invention offers a greatly expanded Surface Enhanced Raman Scattering technique through which coatings may be altered as appropriate for specific substances and through which expanded ranges and sensitivities can now be accomplished. Thus the detection of substances which are controlled, substances of a medical nature, or simply some new types of analytes are now possible. In its approach, the invention breaks with what may have been preconceptions and utilizes a substance, such as diazonium, which is highly reactive with the controlled substance or other analyte at issue. Thus the diazonium and the like may interact to permanently bond or perhaps covalently bond with the analyte as opposed to the more typical weak chemisorption or physisorption. Through this reaction the diazonium may be viewed as an advantage rather than a hindrance in that an actual adduct is created and rather than studying the analyte in isolation the resulting adduct is studied.
In a more basic form, the invention includes a method of detecting molecules by chemically binding them to a surface and then using a surface-sensitive detection method. This is a significant improvement over the existing method of attracting molecules to a surface through weak forces. The new technique increases the sensitivity of detection by forming a strong bond between the analyte and the surface coating. The equilibrium between surface analytes (e.g. detectable analytes) and solution analytes (e.g. not detectable since not captured by the surface) may be characterized by an equilibrium constant. In one sense, the equilibrium constant describes the number of species at the surface relative to solution. In another sense, it describes the rate at which molecules go on the surface relative to the rate at which they come off. The present invention describes a method of forcing the off rate to be essentially zero and to thereby greatly increase the number of species on the surface (e.g. the detectable species). This is one form of sensitivity improvement. A second method of improvement comes from the ability to wash the surface after the analyte has bonded to the surface. In many cases, the sensitivity of an analytical method has been limited due to the presence of large interfering backgrounds. This background arose from species in the solution with the analyte that gave rise to a signal that was similar to that of the analyte. These interfering species can now be removed with this invention since the analyte is bound to the surface and the interfering species can be easily washed off without affecting the analyte. (This, of course may, not work for every analyte possible.) A third method of improvement comes from the ability to distinguish between different analyte types. Because the adduct formed through formation of a covalent bond is a new and unique chemical species, it will likely have unique spectroscopic characteristics. Since different analytes will form different adducts, they may be distinguished from each other on the basis of the differing spectra, thus decreasing the likelihood of mistaken identification.
In the field of sensing controlled substances, it is thus an object of the invention to provide techniques and devices which may be applied to a greater variety of controlled substances and which may be applied to all substances with a higher degree of sensitivity, with greater ability to discriminate, and even with the possibility of confirmational systems to be automatically in place to avoid false indications. In keeping with these objects, it is a goal of the invention to offer a system which is controlled to a lesser degree by the equilibrium constants of a reaction and which even offers situations where the equilibrium constant is essentially zero. Similarly, a goal is to provide a system in which interfering substances and the like can be removed without concern of removing or somehow reducing the signatures available as a result of the controlled substance itself. A further goal is for a system which permanently binds the controlled substance to the sensor surface and thus can be analyzed at any location or any time without a high degree of concern of degradation of the signal.
A more broadly stated goal for the invention in the field of sensing controlled substances is that of providing techniques which can be designed for specific chemicals in specific situations. Thus a goal is to provide a system which can be optimized through chemical design for either specific substances or for specific ranges and broader substances in one analytical event.
An object as it relates to the broader field of analytical chemistry in general, as well as in the field of detecting controlled substances, is to provide a system in which internal standards can permit the monitoring and compensation for alterations in the illumination source as well as to provide techniques in which coatings rather than the surfaces themselves can be used in a variety of detection techniques. Thus, the invention could be used with a large variety of analytical techniques, known to those in the art. Optical techniques such as Raman, fluorescence, or absorption spectroscopy could be used for detection. Mechanical detection would also be possible with sensitive mass sensors. Raman scattering may prove to be one of the most desirable detection methods. Surface Enhanced Raman Scattering (SERS) provides large enhancements in the Raman scattering from molecules near certain metal surfaces. The invention thus involves methods of anchoring our molecule specific probes to this type of surface. Moreover, while the invention contains a fairly high specificity for specific classes of molecules it may not need to be truly specific to a single molecule. SERS, of course, is a truly molecular-specific detection method. This means that a complex mixture of species in a reactive class of compounds could react with the surface and SERS could differentiate and quantitate between the various species.
The use of SERS also provides a substantial improvement over many techniques through the use of an internal standard. The internal standard furnished by this invention can be the portion of the surface tether that is unchanged by the reaction of the reactive functional group (RFG) with the analyte. This portion of the surface coating may give rise to a SERS signal that can be monitored simultaneously with that of the analyte. A simple ratio of the two signals or a more sophisticated multi variate analysis can be used to give a detection method that is independent of source intensity fluctuations or variations in the detection throughput. This creates the possibility for more simplified and less expensive instrument design as well as design for rugged, maintenance free use.